The results from viability assessment of treated cell lines revealed large variations within the cell line panel. Notably, HT appeared to sensitize B76 in treatment with mitomycin and cisplatin. In contrast, viability of OVCA433 was not affected by HT under any treatment, and the cell line appeared to be non-responsive to HT.
OVCA432 appeared most resistant to drugs tested at clinical concentrations, while CaOV3 was hypersensitive to mitomycin and sensitized to cisplatin in combination with HT (fig. 15). Determining the cell cycle distribution and rate of apoptosis within cell lines 24 hours after treatment could help explain the cell-line specific response to combination treatment.
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4.5.1 Cell cycle distribution and apoptosis in pmoc8, B76 and OVCA433 after combination treatment
Cell lines pmOC8, B76 and OVCA433 were fixated both prior to treatment (marked 0 hours), as well as 24 h after treatment. Cells were stained with Hoechst and TUNEL for cell cycle and apoptosis analysis, respectively. Figures represent one biological replicate for pmOC8 and OVCA433, and two replicates for B76.
The highest rate of apoptosis caused by HT alone was found in the B76 cell line, with 27% apoptotic cells 24 h after treatment compared to only 4% in the control (fig. 16A). The other cell lines pmOC8 and OVCA433 had only 13%, but OVCA433 had equal frequency of apoptotic cells in the control independent of temperature (fig.
16C/D). For cisplatin, B76 cells treated at 37ºC had higher fraction in S-phase and 11% apoptosis after 24 hours. However, cisplatin and HT in combination resulted in smaller fraction in S-phase and 37% apoptosis, up 10% from HT alone (fig. 16A). For mitomycin, the fraction of apoptotic cells were 17% and 28% for samples treated at 37ºC and 42ºC, respectively. As such, no net increase in apoptosis could be seen based on 42ºC control. We also included carboplatin, and to our surprise, while no increase in apoptosis were seen at normal temperature, HT caused an increase in apoptosis to 32%, up 5% from HT alone. For B76, a trend was seen; the fraction of cells in S-phase was smaller for HT treated samples compared to samples treated at 37ºC.
For pmOC8, HT alone resulted in 13% apoptosis at 24 h after treatment, compared to 3% in the control (fig. 16B). For pmOC8, cells treated with HT in combination with cisplatin or mitomycin had lesser fraction of cells in S-phase compared to
treatment at 37ºC (fig. 16B). The amount of apoptotic cells for cisplatin was however equal at both temperatures, but comparing the values to the control samples (3% for 37ºC control, 13% for HT), treatment at 37ºC appeared to cause more apoptosis at 24 hours after treatment. The same effect could be seen for mitomycin; 31% apoptosis were found in the sample treated at 37ºC but only 26% in combination with HT.
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Figure 16: Summary of apoptosis and cell cycle distr ibution observed in cell lines after flow cytometry, A) B76, the only cell line were carboplatin treated samples were included, B) pmOC8, and C)
OVCA433. Figure D) shows the apoptosis frequency in control samples for the three cell lines, prior to treatment (0 h) and 24 hours after treatment. Note the high fraction of apoptotic cells in B76, and the thermo-tolerant behavior of OVCA433.
Both pmOC8 and B76 displayed similar trend, as HT appeared to decrease the fraction of cells in S-phase compared to samples treated at 37ºC. We postulated that
47 it could be due to a transitory cell cycle arrest caused by HT, which prevented
initiation of S-phase for cells and possible fixation of crosslink damage.
The OVCA433 cell line had equal rates of apoptosis after treatment at 42ºC and 37ºC (fig. 16C/D), which upheld the thermo-tolerant behavior observed from viability assays. In contrast to the cell lines B76 and pmOC8, OVCA433 had higher fraction of cells in S-phase after treatment at 42ºC compared to 37ºC. The rate of apoptosis found 24 hours after cisplatin treatment at 37ºC were 29%, compared to 22% after treatment at 42ºC. OVCA433 appeared to be sensitive to mitomycin, as 63% cells were apoptotic after mitomycin treatment at 37ºC. Surprisingly, this fraction was lower (39%) in samples treated with mitomycin and HT. While the results from treatment at 37ºC were in compliance with the low viability observed after 72 hours by MTS assay, the lower fraction of apoptotic cells observed after combination treatment with HT could not be explained without additional time-points of analysis.
4.5.2 Cell cycle distribution after cisplatin treatment
The cell line responses detected by Hoechst and TUNEL assay after treatments were surprising. In most cases, drug treatments in combination with HT did not cause increased rates of apoptosis after 24 hours compared to treatments at 37ºC. In contrast to viability assays, where sensitizing effects of HT were observed with cisplatin for both B76 and pmOC8, only B76 showed small increase in apoptosis after HT combination treatment (fig. 16A). To determine if this could be due to specific interactions between HT and cell cycle progression, DNA histograms are shown (fig. 17).
The histogram for B76 showed a similar phenomenon as in pmOC8 (fig. 16A/B). A double-peak at the G1/S transition indicated that cells had attempted to start S-phase but shortly after stalled. It is likely caused by cells encountering cisplatin-induced crosslinks in DNA, making them unable to continue replication until the crosslink is resolved. As this effect is not seen for cisplatin in combination with HT, it is possible that HT induces a transitory G1-arrest in B76 and pmOC8 that
48 prevented S-phase initiation until after 24 hours. However, this hypothesis could only be confirmed by examination of cell cycle distribution at several more time-points after treatment. In contrast, the cell cycle distribution of OVCA433 did not appear to be affected by HT (fig. 17D).
Figure 17: Histograms of DNA and apoptosis measurements from TUNEL assay 24 hours after cell treatment. Figure shows the distribution of cell lines after treatment with cisplatin with or without HT (42ºC). Note the sharp peak immediately following G1 -phase for A) B76 and B) pmOC8 treated with cisplatin at 37ºC. Both cell lines have likely attempted to initiate S -phase but have encountered cisplatin crosslinks, stalling replication. C) OVCA433, on the other hand, has high G2/M -arrest at 37ºC and accumulation in S-phase at 42ºC. Note the increase in apoptosis of A) B76 after treatment with both cisplatin and HT.
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4.5.3 Cell cycle and apoptosis in B76 after 72 hours
To determine if the rate of apoptosis seen in B76 24 hours after HT (27%) was maintained at the duration used for viability assay, we investigated the cell cycle distribution and apoptosis in this cell line 72 hours after treatment. While cell cycle distribution appeared similar, apoptosis detected by TUNEL-assay was 3% and 14%
in normal versus HT treated sample, respectively (figure 18). The fraction of apoptotic cells was 27% after 24 hours and 14% after 72 hours, suggesting the effects of HT on B76 apoptosis can be delayed over 72 hours. For a complete overview over data obtained during flow cytometry analysis, see appendix 5.
Figure 18: Histogram of DNA content as well as apoptosis stained with TUNEL. The B76 control samples (no drug) treated at 37ºC and 42ºC. Cells were analyzed after 72 hours recovery in incubator.
The amount of apoptosis was detected by TUNEL staining (top), note 14% apoptotic cells from HT alone, which decreased from 27% as detected 48 hours earlier.
As the fraction of apoptotic B76 cells treated with carboplatin indicated that the cell line was responsive and not resistant as previously assumed, we analyzed samples
50 treated with carboplatin after 72 hours. To our surprise, the assumed carboplatin-resistant B76 did respond to carboplatin if treated in combination with HT. This sensitizing effect could not be detected by viability assays, where viability was observed to decrease only marginally (fig. 11).
Figure 19: TUNEL assay 72 hours after treatment of B76 with carboplatin and cisplatin. Notice the high fraction of apoptotic cells after combination treatment at 42 ºC for both drugs. Accurate
determination of the apoptotic fractions b ecomes impossible when there is no clear distinction between apoptotic and living cells, as observed in B76 sample treated with cisplatin and HT.
The cell lines pmOC8 and OVCA433 were not analyzed after 72 hours due to time constraints. Analysis of B76 after 72 hours, however, revealed higher apoptotic fractions resulting from combination treatment. Compared to the apoptotic fractions
51 observed after 24 hours, these observations suggest that drug treatment induces apoptosis faster in cells treated at 37ºC. Figures represent one biological replicate.
As samples from B76 after 72 hours incubation contained high amounts of cell fragments (fig. 19), it must be made clear that accurate interpretation of data was difficult and only provided an estimation of the apoptotic fractions.